Cytokinesis in Arabidopsis: Cytokinetic vesicles, cargo proteins and fusion machinery

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In plant cytokinesis, the partitioning membrane (aka cell plate) originates through targeted delivery of secretory vesicles along a dynamic cytoskeletal array (aka phragmoplast) to the cell division plane where these vesicles fuse with one another and later with the margin of the laterally expanding cell plate. The cell plate undergoes maturation including the stepwise synthesis and assembly of the newly forming cell wall. The membrane fusions involve specific Qa-SNAREs (such as KNOLLE or SYP132) which form trans-SNARE complexes with their partners. What cargo proteins are transported by the cytokinetic vesicles and what roles they might play in cytokinesis is virtually unknown. In the current funding period (JU 179/24-1), we have established methodological foundations to identify cargo proteins resident on cytokinetic vesicles, using immunoprecipitation followed by mass spectrometry. Of 250 candidates that we have detected on KNOLLE-tagged vesicles, SYP132-tagged vesicles or both groups, we have selected 17 membrane-associated proteins encoded by G2/M-phase specifically expressed genes for more detailed analysis in transgenic plants.(1) A primary aim will be to address the question of whether there is only a single population of cytokinetic vesicles or whether there are several ones. For example, it is conceivable that vesicles bearing specific SNARE protein complexes transport only specific subsets of cytokinetic cargo proteins. Another possibility could be that newly synthesised cargo proteins are transported on other vesicles than proteins that are endocytosed from the plasma membrane and re-targeted to the plane of cell division.(2) Another aim will be to analyse the functional or structural contributions of cargo proteins – both known ones and those to be identified – to cytokinesis. We will give preference to membrane-associated proteins that are specifically synthesised during G2/M phase of the cell cycle. We will generate knockout mutants and analyse them for possible cytokinesis defects, using suitable subcellular markers or ultrastructural analysis. In addition, we will attempt to identify interacting proteins.(3) We will also pursue new strategies with which to identify cytokinetic cargo proteins. Along these lines, we will attempt to increase the proportion of dividing cells in the starting material for IP-MS analysis. In addition, we will explore a very different approach. We will screen available datasets of genes specifically expressed during G2/M phase for genes that encode membrane-associated proteins and have so called MSA ("mitosis-specific activator") elements in their promoter regions. Again, we will predominantly focus on proteins of unknown function whose sequences have been conserved in evolution.